McLean F M, Keller P J, Genge B R, Walters S A, Wuthier R E
J Biol Chem. 1987 Aug 5;262(22):10481-8.
Studies were made on the disposition of mineral ions in matrix vesicles (MV) and their relationship to alkaline phosphatase by treatment of MV-enriched microsomes (MVEM) with graded levels of Ca2+-chelating agents to complex accessible ions, fractionation of MVEM on hypertonic sucrose gradients at two different pH values (7.5 and 8.0) to evaluate for the presence of calcium phosphate mineral, and passage of MVEM through cation-exchange columns to determine the accessibility of the Ca2+. The effect of removal of Ca2+ and Pi on subsequent ability of MVEM to induce mineral formation from synthetic cartilage lymph was also determined. Passage through cation-exchange columns revealed that MV Ca2+ was not freely exchangeable, but coeluted in the void volume with alkaline phosphatase. However, upon incubation in synthetic cartilage lymph, progressively more Ca2+ was retained by the column. These findings indicate that, initially, the majority of Ca2+ in MVEM is internal and not readily exchangeable, but as Ca2+ accumulates, progressively more becomes external. The mineral in MV is labile and readily susceptible to loss; treatment with graded levels of EGTA removed major portions of the original Ca2+ and Pi. 45Ca uptake by these mineral-depleted MV was markedly reduced, even in the presence of alkaline phosphatase substrates. Sucrose gradient fractionation of MVEM caused extensive loss of Pi, but not Ca2+, from the low-density alkaline phosphatase-rich fractions. This reveals that Ca2+ and Pi are not initially coupled together: Pi is largely soluble, whereas Ca2+ must be tightly bound. In the high-density vesicles, large amounts of both Ca2+ and Pi are present. The slightly enhanced recovery at higher pH suggests the presence of a solid mineral phase. During mineralization by MV, Ca2+ became externalized, and concomitantly alkaline phosphatase activity declined. This suggests that a direct association exists between the enzyme and the developing mineral.
通过用不同浓度的Ca2+螯合剂处理富含基质小泡的微粒体（MVEM）以络合可及离子，在两种不同pH值（7.5和8.0）下在高渗蔗糖梯度上对MVEM进行分级分离以评估磷酸钙矿物质的存在，以及使MVEM通过阳离子交换柱以确定Ca2+的可及性，从而研究了基质小泡（MV）中矿物质离子的分布及其与碱性磷酸酶的关系。还确定了去除Ca2+和Pi对MVEM随后从合成软骨淋巴诱导矿物质形成能力的影响。通过阳离子交换柱的实验表明，MV中的Ca2+不能自由交换，而是与碱性磷酸酶一起在空体积中被洗脱。然而，在合成软骨淋巴中孵育后，柱中保留的Ca2+逐渐增多。这些发现表明，最初，MVEM中的大部分Ca2+是内部的且不易交换，但随着Ca2+的积累，越来越多的Ca2+变为外部的。MV中的矿物质不稳定且容易丢失；用不同浓度的乙二醇双四乙酸（EGTA）处理会去除大部分原始的Ca2+和Pi。即使存在碱性磷酸酶底物，这些矿物质耗尽的MV对45Ca的摄取也明显减少。MVEM在蔗糖梯度上的分级分离导致低密度富含碱性磷酸酶的部分大量丢失Pi，但不会丢失Ca2+。这表明Ca2+和Pi最初并未结合在一起：Pi大部分是可溶的，而Ca2+必须紧密结合。在高密度小泡中，同时存在大量的Ca2+和Pi。在较高pH值下回收率略有提高表明存在固体矿物质相。在MV矿化过程中，Ca2+外化，同时碱性磷酸酶活性下降。这表明该酶与正在形成的矿物质之间存在直接关联。
